Heme proteins such as hemoglobin and myoglobin play an essential role in stabilizing molecular oxygen for transport and storage. The oxygen carrying portion of the red blood cell is hemoglobin, a tetrameric protein molecule composed of two identical alpha globins (alpha 1, alpha 2), two identical beta globins (beta 1, beta 2) and four heme molecules. A heme molecule is incorporated into each of the alpha and beta globins to give alpha and beta subunits. Heme is a macrocyclic organic molecule that contains an iron atom at its center; each heme can combine reversibly with one ligand molecule, for example oxygen. In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form two stable alpha/beta dimers, which in turn associate to form the tetramer (a homodimer). The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges. Ligands, particularly oxygen, bind reversibly to the reduced form of the iron (ferrous, Fe 2+) in the heme. Other ligands which compete with oxygen for the heme group include carbon monoxide and nitric oxide.
It is not always practical to transfuse a patient with donated blood. The well known complications of blood transfusion namely incompatibility reactions, disease transmission, immunosuppression and the storage limitations of erythrocytes points to the need for the development of blood substitutes devoid of these shortcomings. In these situations, use of a red blood cell substitute is necessary. A “blood substitute” is a preparation that does not necessarily replace blood in all of its functions, but an emergency resuscitative fluid that is capable of efficiently transporting oxygen to tissue. This fluid, however, must be free of toxic side-effects, as well as of agents of disease such as bacteria and viruses.
For over 50 years, efforts directed to the development of a blood substitute have focused on hemoglobin (Hb). Hemoglobin (Hgb) is the oxygen-carrying component of blood. Hemoglobin circulates through the bloodstream inside small enucleate cells called erythrocytes (red blood cells). Hemoglobin is a protein constructed from four associated polypeptide chains, and bearing prosthetic groups known as hemes. The erythrocyte helps maintain hemoglobin in its reduced, functional form. The heme iron atom is labile to oxidation, but may be reduced again by one of two enzyme systems within the erythrocyte, the cytochrome b5 and glutathione reduction systems.
Hemoglobin exhibits cooperative binding of oxygen by the four subunits of the hemoglobin molecule (two alpha-globins and two beta-globins in the case of HbA), and this cooperativity greatly facilitates efficient oxygen transport. Cooperativity, achieved by the so-called heme—heme interaction, allows hemoglobin to vary its affinity for oxygen. Hemoglobin reversibly binds up to four moles of oxygen per mole of Hb. At high oxygen concentration, such as that found in the lungs, the oxygen affinity is high and hemoglobin is almost saturated with oxygen. At low oxygen concentration, such as that found in actively respiring tissue, the oxygen affinity is lowered and oxygen is unloaded. The oxygen affinity of hemoglobin is lowered by the presence of 2,3-diphosphoglycerate (2,3-DPG), chloride ions and hydrogen ions. Respiring tissue releases carbon dioxide into the blood and lowers its pH (i.e. increases the hydrogen ion concentration), thereby causing oxygen to dissociate from hemoglobin and allowing it to diffuse into individual cells.
The ability of hemoglobin to alter its oxygen affinity, increasing the efficiency of oxygen transport around the body, is dependent on the presence of the metabolite 2,3-DPG. Inside the erythrocyte 2,3-DPG is present at a concentration nearly as great as that of hemoglobin itself. In the absence of 2,3-DPG “conventional” hemoglobin binds oxygen very tightly and would release little oxygen to respiring tissue.
Aging erythrocytes release small amounts of free hemoglobin into the blood plasma where it is rapidly bound by the scavenging protein haptoglobin. The hemoglobin-haptoglobin complex is removed from the blood and degraded by the spleen and liver.
It is clear from the above considerations that free native hemoglobin A, injected directly into the bloodstream, would not support efficient oxygen transport about the body. The essential allosteric regulator 2,3-DPG is not present in sufficient concentration in the plasma to allow hemoglobin to release much oxygen at venous oxygen tension, and free hemoglobin would be rapidly inactivated as an oxygen carrier by auto-oxidation of the heme iron.
Therefore, a need exists for a substitute other than hemoglobin which can bind and carry oxygen to cells. This substitute may also be used in other applications where hemoglobin is used, including as a biological sensor for oxygen. The present invention provides proteins which meet that need.